Contraction of fast vertebrate skeletal muscle is closely controlled by calcium fluxes between intracellular compartments, which in turn are controlled by the membrane potential. Many specific features of the regulatory mechanisms for excitation-concentration coupling are unclear. The proposed research will utilize single amphibian muscle fibers, microdissected to give access to the intracellular space and membranes, dor direct assay of 45Ca efflux, 45Ca influx, and related variables, under controlled conditions in situ. Microdissection methods that may have differing effects on transverse invaginations of the cell membranes will be compared. Stimulated calcium efflux from intracellular stores i the sarcoplasmic reticulum to the myoplasm will be analyzed with respect to dependence on nucleotide and on imposed voltage gradients across the sarcoplasmic reticulum membrane, assessed from the Nernst distribution of radioactive probe ions. The calcium influx mediated by the active calcium transport system of the sarcoplasmic reticulum will be analyzed with respect to magnesium ion concentration and depolarizing conditions. Both fluxes will be analyzed with respect to the effects of drugs and other agents known to inhibit or stimulate net or unidirectional fluxes in intact muscle and/or isolated sarcoplasmic reticulum vesicles. These studies relate directly to elucidation of the physiological control of the sarcoplasmic reticulum by cell membrane potential, of the excitatory signal to the reticulum, of reticulum membrane mechanisms that mediate calcium efflux, and of the operation of the active calcium transport system in situ. They have immediate relevance to the normal and pathological function of skeletal muscle and implications for the function of cardiac and smooth muscle, as well as the basis of pharmacological effects. Moreover, the central steps in excitation-contraction coupling constitute a model for the regulation in other cell types of functions that are controlled by intracellular calcium movements.